U.S. Pat. No. 6,594,011 issued Jul. 15, 2003, the entirety of which is incorporated by reference herein for all purposes, discloses an imaging apparatus and method for real time imaging ellipsometry for high throughput sensing of binding events useful in molecular interaction analysis including biotech applications. The apparatus disclosed employs the immobilization of an array of binding or capture molecules (“ligands”) on a planar surface of a transparent substrate and the use of a beam of polarized light directed at the underside of the surface in a manner to achieve total internal reflection (TIR) and generate an evanescent field in the plane of the ligands. The ligands are exposed to a biological sample and analytes in the biological sample bind to different patterns of the immobilized ligands in a manner to change the polarization at locations in the array at which binding occurs. An image of the array is compared with a stored image of the initial light polarization shifts to determine the location and magnitude of binding events within the array, thus identifying and quantitating the analytes present in the biological sample.
The apparatus for implementing the foregoing technique employs a prism or gratings to achieve the requisite TIR generated evanescent field, the prism being the most practical implementation for imaging applications.
TIR imaging ellipsometry works well for fields of view up to 1-2 cm2, which permits real time imaging of tens of thousands of binding events simultaneously. However, there is a need to be able to image or scan areas which are much larger, such as 128 mm×86 mm (e.g., the area of both 384 well and 96 well plates) to permit lower costs per test and for multiple tests per patient for large numbers of patients simultaneously, which is increasingly a requirement for clinical diagnostics and personalized medicine. Simply scaling up the prism geometry so that the field of view covers an entire 1536 well, 384 well or 96 well plate has the following practical and technical drawbacks. In addition to the high expense of large optics, image quality becomes more challenging to maintain as the field of view is increased. Spherical aberration effects, optical path-length and depth of field issues demand much more space in order to keep the quantitative imaging errors low. Additionally, because the optics are too expensive and bulky to be part of the disposable plate, the optical coupling to the disposable plate is done by the user and in many applications, this is not practical, especially when high throughput is required. Obviating the need for a prism simplifies both the instrument and interface between the instrument and disposable multiwell plate.